Genome-wide analysis of annexin gene family in Schrenkiella parvula and Eutrema salsugineum suggests their roles in salt stress response

Annexins (Anns) play an important role in plant development, growth and responses to various stresses. Although Ann genes have been characterized in some plants, their role in adaptation mechanisms and tolerance to environmental stresses have not been studied in extremophile plants. In this study, Ann genes in Schrenkiella parvula and Eutrema salsugineum were identified using a genome-wide method and phylogenetic relationships, subcellular distribution, gene structures, conserved residues and motifs and also promoter prediction have been studied through bioinformatics analysis. We identified ten and eight encoding putative Ann genes in S. parvula and E. salsugineum genome respectively, which were divided into six subfamilies according to phylogenetic relationships. By observing conservation in gene structures and protein motifs we found that the majority of Ann members in two extremophile plants are similar. Furthermore, promoter analysis revealed a greater number of GATA, Dof, bHLH and NAC transcription factor binding sites, as well as ABRE, ABRE3a, ABRE4, MYB and Myc cis-acting elements in compare to Arabidopsis thaliana. To gain additional insight into the putative roles of candidate Ann genes, the expression of SpAnn1, SpAnn2 and SpAnn6 in S. parvula was studied in response to salt stress, which indicated that their expression level in shoot increased. Similarly, salt stress induced expression of EsAnn1, 5 and 7, in roots and EsAnn1, 2 and 5 in leaves of E. salsugineum. Our comparative analysis implies that both halophytes have different regulatory mechanisms compared to A. thaliana and suggest SpAnn2 gene play important roles in mediating salt stress.


Introduction
Salinity is one of the most important abiotic stresses that affects the growth and crop yields, significantly. Soil salinity affects around 50% of arable land worldwide [1], while most food crops are salt sensitive (glycophyte), which poses a threat to global food security. Halophytebased researches can provide valuable information to improve abiotic stress tolerance in crops.
To cope with high salt concentration, Halophytes have evolved mechanisms such as dormancy-like states to salt inhibit germination, improved ion homeostasis, morphological features (e.g., higher stomatal density and a second layer of endodermis), an up-regulated antioxidant system and also "stress ready" transcriptome and metabolome [4]. Annexin (Ann) is a Ca 2+ and membrane-binding protein which is reported to be involved in plant growth, development and various stress responses such as salt stress [5]. The Ann genes have already been discovered in Brassica species such as A. thaliana [6], B. juncea [7], B. rape [8], B. oleracea [9], B. napus [9] and other plants like Oryza sativa [10], Triticum aestivum [11], Zea mays [12], Solanum lycopersicum [13]. A multi-gene and multi-functional family of Ann proteins are involved in important biological activities, including ion transport, cellular homeostasis, membrane trafficking and cytoskeletal organization [14]. Anns as Ca 2+ binding proteins, act as Ca 2+ signal transducers. Laohavisit et al. [15], demonstrated that A. thaliana Annexin1 (AtAnn1) as a ROS-activated plasma membrane Ca 2+ channel, is one of the candidate channel that involves in mediating [Ca 2+ ]cyt accumulation under salinity stress. Apart from AtAnn1, Huh et al. [16] showed that AtAnn4 also interact with AtAnn1 and regulate growth and viability under saline and drought conditions. In rice the expression levels of OsAnn6 and OsAnn7 increased in response to salt stress, while the expression levels of OsAnn1 and OsAnn10 decreased significantly [10]. Yadav et al. [8], discovered that the transcript levels of Anns (BraAnn1, 2,4 and 8) in Brassica rapa increased under salt stress.
Several functional analyses have revealed that Ann genes have a positive effect on plant stress tolerance. AtAnn1 gain-of-function mutants in A. thaliana were more drought tolerant than AtAnn1 loss-of-function mutants [17]. In rice, overexpressing of OsAnn3 reduces water loss by enhancing root length and stomata closure in an ABA-dependent manner and accordingly confers stress tolerance [18]. Also, overexpression of StAnn1 in potato increased drought tolerance in addition to modifying redox state and phytohormone mediated pathways [19]. In addition, overexpression of Ann2 from Solanum pennellii improved salt and drought tolerance in Solanum lycopersicum [20].
The regulatory mechanisms of plant Anns in abiotic stress have been investigated in several studies. In Arabidopsis, under salt stress the SOS2-SCaBP8 complex generates and fine-tunes an AtAnn4-dependent calcium signature [21]. Also, cold-activated OST1/SnRK2.6 phosphorylates AtAnn1 and enhances its Ca 2+ transport activity which generates a Ca 2+ signal that mediates freezing tolerance [22]. In A. thaliana, cryptochrome 2 represses the functions of AtAnn2 and AtAnn3 by affecting their subcellular localization and transmembrane Ca 2+ flow in drought stress [23].
However, there has not been any report on Anns in extremophiles species. A comparative study of the Ann gene family in halophyte models versus A. thaliana as a glycophyte would provide a starting point for understanding how the Ann gene family contributes to the halophyte's salt tolerance. In this study, Ann genes were identified in two halophytes of S. parvula and E. salsugineum through genome-wide analysis. We compared SpAnn and EsAnn gene's structures, conserved motifs and promoter region with A. thaliana's Ann genes to investigate their roles in the salt tolerance of the halophytes. Furthermore, the expression of chosen Ann genes in S. parvula and also E. salsugineum in response to salt stress were investigated. Our findings suggest the roles of SpAnn2 gene in mediating salt stress and provide valuable information for further study on the function of Ann genes in halophyte growth, development and stress responses.

Plant materials and NaCl treatment
Seeds of S. parvula (Lake Tuz ecotype) were sown on a 7:2:1 soil mixture (peat moss/vermiculite/perlite) and stratified for 7 days at 4˚C in dark condition. Then, the seeds were grown at 22/20˚C (day/night) with a relative humidity of 60% and a photoperiod of 16 /8 h (light/dark) at light intensity 130 μmol m -2 s -1 . Three weeks after germination (vegetative development), plants were treated with a 200 mM NaCl solution and water as a control. The shoots of treated and non-treated plants were collected at 3 h, 6 h and 12 h after NaCl treatment. Liquid nitrogen was used to freeze all samples before storing at -80˚C for future use.

Identification and phylogenetic analysis of Ann family members
The TAIR database (http://www.arabidopsis.org/) was utilized to get the Ann protein sequences of A. thaliana. The sequence protein of S. parvula and E. salsugineum were obtained from phytozome v13 (https://phytozome-next.jgi.doe.gov/), EnsemblPlants (https://plants. ensembl.org/index.html/) and Thellungiella (http://thellungiella.org/) by the BLASTP program in which E-value cut-off of 1E-5 were applied [24]. Furthermore, sequence identity >75% and similarity >80% were considered as threshold to determine homology. Finally, sequences with low degrees of similarity, poor domain coverage and proteins with different functional annotations were filtered. The ProtParam tool from Expasy (http://us.expasy.org/tools/protparam) was applied to physiochemically describe the investigated proteins with default settings. Also, to predict protein subcellular localization, the CELLO online program v2.5 was used (http:// cello.life.nctu.edu.tw/) [25].
The protein sequences of the Ann gene family in A. thaliana, S. parvula, E. salsugineum and also B. napus and B. rapa as Brassicaceae species, were aligned by the ClustalW program of MEGA-X [26] by default settings. The bootstrap value for the neighbor-joining method of MEGAX, which was used to construct the phylogenetic trees, was set to 1000 [10].

Gene structure and conserved motif analysis
In order to prediction of the exon and intron structures of Ann genes, the Gene Structure Display Server 2.0 (GSDS v2.0) was used (http://gsds.gao-lab.org/) [

Cis-acting elements and transcription factor binding sites analysis
The PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) was applied to look for cis-elements in the 2 Kb upstream of the 5 0 -UTR of A. thaliana, S. parvula and E. salsugineum Ann genes [32]. The promoter regions were divided into three groups: proximal, median and distal, which were 500, 501-1000 and 1001-1500 bp upstream, respectively. To identify the putative transcription factor binding sites (TFBSs) on promoters, the TF Binding Site Prediction program in the PlantRegMap (http://plantregmap.gao-lab.org/binding_site_prediction.php) was used with a threshold p-value � 1e-6 (apart from SpAnn7 with 381 bp upstream) [33]. PANTHER (http://pantherdb.org/) was used to find the Gene Ontology biological process analysis of the three plants [34].

Nonsynonymous/ Synonymous substitution ratio (Ka/Ks)
The nonsynonymous (Ka) and synonymous (Ks) substitution rates and the Ka/Ks value between homologous gene pairs were calculated using DnaSP v6 [35].

Gene expression analysis of the Ann genes under salt stress
A total RNA extraction Mini Kit (Favorgen1) was used to extract total RNA from the shoots of three-week-old plants (4 to 6 true leaves). RNase-Free DNase I (Yekta Tajhiz Azma1) was used to remove DNA contamination from RNA samples according to the manufacturer's instructions. The Easy cDNA Synthesis Kit (Parstous1) was used to synthesize first-strand cDNA from two μg of total RNA. The reactions were performed using the RealQ Plus 2x Master Mix Green (Ampliqon1) in a qRT-PCR system (CFX96 Dx Real-Time PCR Detection Systems (Bio-Rad)) according to the instructions given by the manufacturer. The primers are listed in S5

Statistical analysis
Three biological replicates were used in all experiments. Data were analyzed via GLM procedure of SAS software version 9.1 (SAS Institute 2003). In order to determine significant differences between mean of the treatments, Tukey's multiple comparisons test (Tukey 0.05 ) was applied.

Identification and phylogenetic analysis of Ann gene family in S. parvula and E. salsugineum
Arabidopsis Ann (AtAnn) proteins were retrieved from the TAIR database and the BLASTP program was used against two databases (Phytozome and Thellungiella for S. parvula, Phytozome and Ensembl Plants for E. salsugineum) to identify putative homologous proteins. Ten and eight Ann genes were identified in S. parvula and E. salsugineum, respectively. Some basic properties of Ann genes are shown in Table 1 have four annexin repeats except SpAnn4-1, SpAnn4-2 and also EsAnn4 which have two annexin repeats. Isoelectric points (pI) extended in the range of 4.91 to 9.56 and from 5.11 to 9.61 for SpAnns and EsAnns, respectively. SpAnns and EsAnns are present in different subcellular locations, including cytoplasm, nuclear and mitochondria. SpAnn2 and EsAnn7 are located in the cytoplasm while SpAnn4-1, SpAnn5 and EsAnn4 are located in the nuclear. Most of the SpAnns and EsAnns were localized in two subcellular organelles. SpAnn and EsAnn 1, 3, 8 as well as EsAnn2, SpAnn2-4 and SpAnn6 are found both in the cytoplasm and nuclear compartments. EsAnn5 is located in nuclear and mitochondria while EsAnn6 is located in the cytoplasm as well as mitochondria. Ann genes are unevenly located on five chromosomes (ch) and five scaffolds in S. parvula and E. salsugineum, respectively. S. parvula has four Ann genes on ch4 (SpAnn3-1, 3-2, 4-1 and 4-2) and two on ch6 (SpAnn6 and SpAnn8) while ch1, 2 and 5 each have only one SpAnn gene. Scaffold 2 and scaffold 10 with three (EsAnn6, 7 and 8) and two (EsAnn3 and 4) respectively, contained the highest number of Ann genes in E. salsugineum. In contrast, scaffold 23, 6 and 9 each have only one EsAnn gene. As the SpAnn7 gene is unlocated, the above-mentioned analyses were not considered for it (Table 1).
In order to clarify the relationships among the Ann proteins from A. thaliana, S. parvula, E. salsugineum and also B. napus and B. rapa as Brassicaceae species, a phylogenetic tree was constructed by aligning protein sequences in the MEGA-X. The results indicated that Ann protein family could be classified into six groups (group I to VI). All AtAnn genes were identified to have orthologous genes in S. parvula and E. salsugineum. According to these findings, Ann1, Ann2, Ann5 and Ann8 are clustered in groups III, II, IV and VI, respectively. Meanwhile, Ann6 and Ann7 are clustered in group I, while Ann3 and Ann4 belong to group V (Fig 1).

Gene structure and conserved motif analysis of SpAnn and EsAnn
The SpAnn and EsAnn gene family structures were analyzed and drawn using GSDS v2.0. In the two studied halophytes and A. thaliana, EsAnn6 and AtAnn5 have the longest and shortest length of genomic sequence, respectively. The results showed that the majority of homologous Ann gene pairs in three studied plants, had the same gene structure. According to these findings, the number of exons per gene ranged from 3 to 6 (regardless of SpAnn7). Gene structures are comparable among members of the same phylogenetic group. Six exons and five introns are found in three groups: group IV (Ann5), group V (Ann3 and Ann4) and group VI (Ann8). Group II (Ann2) have five exons and four introns and also group I (Ann6 and Ann7) have four exons and three introns. In group III, a little difference was observed in which Ann1 in A. thaliana has 3 exons and two introns, but EsAnn1 and SpAnn1 have 5 exons and 4 introns in their structure. This data suggests that all of these Ann genes have a common ancestor gene and because of their role in development and many biological processes, they have been shown to be conserved during plant evolution (Fig 2).
All of the Ann proteins, regardless of SpAnn7, were subjected to multiple sequence alignment (MSA) and then the results were used to identify several conserved motifs and residues. Repeats I and IV include Ca 2+ binding sites (GXGT-38-D/E). All Anns have a conserved tryptophan residues except Ann4 and Ann5, which is required for membrane binding. Total Anns except Ann3 and Ann4 in all studied plants, contain the key peroxidase residue (His40). In repeat IV, each Ann contains 'DXXG,' a putative GTP-binding motif, although Ann8 and Ann4 in all investigated plants have 'EXXG' and 'KXXG,' respectively, instead of 'DXXG' (SpAnn4-2 has NXXG). In AtAnn5, nonpolar Glycine residue replaced by polar Serine residue in 'DXXG'. Twelve out of 25 studied Anns in three studied plants, have 'IRI', an F-actin binding motif, in their repeat III but it changed to 'IRV' in AtAnn8 and SpAnn8 while it changed to 'ISV' in EsAnn8. The 'IRI' changed to 'IQI' and 'LYI' in Ann5 and Ann3, respectively (Fig 3).
We investigated conserved motifs in 25 Anns of three studied plants to make it easier to compare Ann families. A total of five motifs with 11 to 41 residues were discovered and then were annotated by InterProScan. Motif1 was found in all four annexin repeats as a core sequence and motif5 found in the fourth annexin repeats near the C-terminus (S1 Table). Group I (Ann6 and 7) and group VI (Ann8) members have a similar motif structure in three plants investigated. In group II, AtAnn2 was different from SpAnn2 and EsAnn2 in which the first annexin repeat is missing motif4 in the C-terminus, whereas the second annexin repeat in AtAnn2 has additional motif2 and its fourth annexin repeat is missing motif2 in the N-terminus. In group III, At and SpAnn1 have a similar motif structure but EsAnn1 lacks motif2 in its N-terminus of second annexin repeat. In group IV, EsAnn5 and SpAnn5 have a similar motif structure but AtAnn5 lacks motif3 in its C-terminus of the second annexin repeat and it has an extra motif2 in its N-terminus of the third annexin repeat. The greatest difference was detected in group V. The members of this group have different motif structures in Ann4 and all of them have two core sequences of annexin repeats. AtAnn3 and EsAnn3 have structures similar to Sp4g21120 (SpAnn3-1) but differ from Sp4g21140 (SpAnn3-2) in that it lacks motif4 in the C-terminus of the first annexin repeat and has an extra motif2 in the second annexin repeat (Fig 4).

Analysis of cis-acting elements and transcription factor binding sites in SpAnn and EsAnn gene promoters
As shown in Fig 5, the promoter of the studied Ann genes contains several types of stress responsive elements, including LTR (low temperature responsive elements), DRE1  Table). AtAnns, EsAnns and SpAnns contain cis-acting elements involved in phytohormone responses such as ABRE4, ABRE3a and ABRE (ABA responsiveness elements); TCAelement, TCA and SARE (salicylic acid responsive elements); TGA-element, TGA-box and AuxRR-core (auxin responsive elements); CGTCA-motif and TGACG-motif (MeJA responsive elements); TATC-box, GARE-motif and P-box (gibberellin responsive elements). This data indicates that Ann genes are highly regulated by multiple cis-acting elements during growth and may play a key role during stress responses.
The results also revealed the presence of several light responsive cis-elements in the Ann promoter region of three studied plants. Also, ABA and MeJa related cis-acting elements had the largest number of hormone-responsive cis-elements, respectively. The MeJA-cis-acting elements found in all members of Ann gene family except for EsAnn1, EsAnn6 and AtAnn8. All the Ann gene promoters contain ABRE except AtAnn6 and SpAnn8 (Fig 5). In our study, SpAnn1 had the largest number of ABRE and also ABRE3a and ABRE4 were more in two halophyte Ann genes compared to A. thaliana. The results also showed that two halophytes have more MYB and Myc cis-acting elements than A. thaliana. All these data clearly suggest that some Ann genes expression may be regulated by salt stress conditions.
To obtain information about transcription factors (TFs) of Ann genes in three studied plants the putative promoter region of each Ann genes was analyzed and various TF families, including AP2/ERF, DOF, GATA, MYB, bZIP and etc. were identified (S3 Table). Additionally, TFBS in the promoter region of studied Ann genes were different in terms of number and distribution. For instance, 11 different TFBSs were found in EsAnn5, while EsAnn4, AtAnn4, and AtAnn6 have only one type of TF binding site. Our results showed that AtAnn5, EsAnn5 and SpAnn8 have the largest number of TFs and also AtAnn4, EsAnn4 and SpAnn6 have the least number of binding sites in each studied plant. The largest number of binding sites belongs to BBR-BPC which is followed by DOF, while CCCH zinc finger (C3H) in EsAnn2 and Zinc Finger HomeoDomain (ZF-HD) in SpAnn1 with only one binding site have the least number of TFs. Interestingly, the number of GATA TFs found in the SpAnn2 promoter of S. parvula (28 GATA TFs) was larger than the total number of GATA TFs found in E. salsugineum (16 GATA TFs) and also the total number found in A. thaliana (13 GATA TFs). It is noteworthy that, there are six bHLH TF binding sites in Ann2 of both halophytes, but none in Ann2 of A. thaliana (Fig 6). This indicates that members of the GATA, DOF and bHLH transcription factor families could possibly regulate the Ann gene in extremophile plants, which needs further experimental verification.

Nonsynonymous/Synonymous substitution rate ratio (Ka/Ks)
The evolutionary constraints affecting the Ann gene family were determined using the Ka, Ks, and the Ka/Ks value. Differences between the aligned sequences may result in differences in amino acids (nonsynonymous changes) or leave the amino acids unchanged (synonymous changes) and counting them up will provide us an idea of how much the sequence has been changed. The ratio of the number of nonsynonymous substitutions per nonsynonymous site (Ka) to the number of synonymous substitutions per synonymous site (Ks) determine the selective forces acting on the protein. The Ka/Ks value can be greater than one, less than one or equal to one, indicating positive, purifying or neutral selection, respectively. Positive selection fixes beneficial variations, while purifying selection removes deleterious variations, whereas neutral selection is neither beneficial nor detrimental on a set of homologous proteincoding genes [40,41]. The majority of Ann genes had a Ka/Ks value less than one, which implies that these genes during the evolution have been subjected to purifying selection. Nevertheless, the Ka/Ks value of AtAnn2/SpAnn2 which was greater than one signifies positive selection (S4 Table).
Based on cis-acting elements and TFs analysis as well as previous studies, three Ann genes (SpAnn1, 2, and 6) in S. parvula were selected to confirm the in-silico results using qRT-PCR. We found that compared to the control, the studied genes were up-regulated at least at one of the salt treatment time points (P�0.05). In comparison with control conditions, in 6 h after salt stress, SpAnn1 gene expression was significantly up-regulated (with 2.20 relative expression level). SpAnn2 gene expression was highly induced 3h after salt stress (with 4.17 relative expression level) and significantly repressed 6 and 12 h later (with 1.16 and 1.44 relative expression level respectively). SpAnn6 gene expression was significantly increased 3 h after salt stress (with 2.13 relative expression level) compared to 6 h and 12 h (Fig 8). Analysis of candidate gene expression (SpAnn1, 2, and 6) in root of S. parvula was retrieved from Li et al.
[39] (S7 Table) research. Based on the results, SpAnn6 showed the greatest difference in 3 h after salt treatment. However, weak changes (range from -0.447 to +0.496) was detected in root (S2 Fig). These findings imply the role of mentioned genes in the salt stress response in the shoot of S. parvula (Fig 8).

Discussion
Plants are exposed to various abiotic stresses, which influence crops growth and development. Ann gene family are considered to play important role in abiotic stress tolerance [5,14]. Several Ann genes have been discovered in plants, including eight in Arabidopsis [6], ten in rice [10], 25 in wheat [11] and 26 in B. napus [9] but up to now there hasn't been reported any study about the Ann gene family in extremophile plants. In this study, eight and ten Ann genes found in the E. salsugineum and S. parvula genomes, respectively. Based on the studies of Oh et al. [42] who worked on genome structural variations in A. thaliana and S. parvula, it was found that shared tandem duplication happened in SpAnn3-1 and SpAnn3-2 and also SpAnn4-1 and SpAnn4-2. The theory that gene duplications often occur as tandem duplications is supported by the similar amino acid sequences, same chromosome localization and short distance between these predicted genes [43] and gene duplication events may have increased the number of Ann genes in S. parvula which is in agreement with Clarks et al. [44] findings. Ann genes were found on five chromosomes and five scaffolds in S. parvula and E. salsugineum, respectively. We were unable to map genes into the chromosomes of two extremophile plants due to limitations in sequencing quality and assembly methods. These proteins were classified into six groups (Fig 1), which was consistent with a previous report on the Brassicaceae species [9]. One of the key factors related to Ann protein's functions is its subcellular distribution profile [14]. Plant Ann proteins are located in different sites, including cytoplasm and plasma membrane [12,45], nucleus [20,46], chloroplast [47] and tonoplast [48]. EsAnns and SpAnns were predicted to have three subcellular localization sites: the cytoplasm, nucleus and mitochondria. Our findings are in partial agreement to those obtained before.
In our study, the majority of homologous Ann gene pairs in three studied plants, had similar gene structures. All 8 members of Ann genes in A. thaliana were identified to have orthologous genes in S. parvula and E. salsugineum. The presence of some conserved motifs and the four repeats in the At, Sp and Es Ann amino acid sequences (Figs 3 and 4), suggests that all of these Ann genes have a common ancestor gene. plant Ann have some conserved motifs and residues which are important for its activity including Ca 2+ binding site, conserved tryptophan for membrane binding, His40 as key peroxidase residue, ATP/GTP binding motif and F-acting binding motif. The findings agree with previous researches [9,10,49] which show that SpAnns and EsAnns are members of a multi-gene family of Anns that are conserved due to their important biological functions. However, there are some differences between the two halophytes and A. thaliana as a glycophyte, which might be linked to their tolerance to abiotic stresses. For instance, comparing AtAnn2 with SpAnn2 and EsAnn2 shows that due to the replacement of Alanine 85 by proline 85 in AtAnn2 (green arrow 1), the first annexin repeat misses motif4 in the C-terminus and the second annexin repeat has additional motif2. Moreover, the fourth annexin repeat in AtAnn2 misses motif2 in the N-terminus through the replacement of Alanine 254 by Serine (green arrow 2) (S1 Fig). He et al. [9], investigated the Ka/Ks value between paralogous Ann gene pairs in A. thaliana,  B. rapa, B. oleracea and B. napus. They found that most of the genes experienced purifying selection but gene pair of BrAnn2-2/BnaAnn2A-2 (Bra024346/BnaA06g23960D) experienced positive selection. In our investigation, AtAnn2/SpAnn2 experienced positive selection whereas the others experienced purifying selection.
Based on cis-acting elements analysis, responses to many biotic and abiotic stresses are associated with motifs found in the promoter regions of the Anns, including ABRE, MBS, DRE1, LTR, MYB, MYC, STRE, TC-rich repeats, as-1, ERE and WUN-motif, etc. (Fig 5). The most abundant cis-elements were the light responsive, which were followed by MYC and ABRE. Furthermore, ABREs were the most numerous among hormone responsive cis-elements. Among the stress related elements, ABRE, ABRE3a, ABRE4, MBS, DRE1, Myb, MYB, Myc, MYC are supposed to be associated with plants' responses to salt stress [50]. It is to be noted that the promoter regions of the two halophytes exhibit a greater number of MYB, Myc and ABRE cis-elements than A. thaliana as a glycophyte. The results suggest that Anns expression may be controlled by hormones, abiotic and biotic stress conditions which implies its role in growth, development and stress tolerance.
In this study, only Ann1, in all three investigated plants, had DRE1 in its promoter. Previous studies supported its role in abiotic stresses [51,52]. Furthermore, all mentioned Ann1 had numerous ABRE that have been shown to improve abiotic tolerances [53][54][55]. Among all the studied Anns, only SpAnn1 had a Zinc Finger Homeo Domain (ZF-HD) and it also had a NAC TF binding site in its promoter, which its role in drought and salt tolerance has been confirmed [56][57][58]. According to cis-acting elements analysis in our study, the expression patterns of selected SpAnn1 as well as EsAnn1 were investigated, which showed their upregulation in response to NaCl treatment. The results imply Ann1's role in abiotic stress tolerance, which is compatible with previous studies [19, 45,59].
Transcription factors (TFs) participate in regulating a wide variety of target genes that are responsible for plant adaptation and tolerance [60]. A variety of TF families, including AP2/ ERF, NAC, WRKY, MYB and bZIP, have been reported to be linked to stress responses [50]. More than 1500 TFs were identified in the Arabidopsis genome sequence [61] and among these TFs, HSFA2, bZIP24, WRKY33, MYB41, ANAC042 and C2H2-type zinc fingers have been suggested to play critical roles in response to salt and osmotic stress [62]. We investigated the regulation of TFs under the expression of Ann genes in A. thaliana, E. salsugineum and S. parvula and found some differences in the TF binding site types, number and distribution (Fig 6). The two halophytes have a greater number of DNA binding with one finger (DOF) compared to A. thaliana. Earlier studies verified DOF involvement in salt tolerance [63]. According to the genes formerly mentioned in E. salsugineum [38], it could be deduced that Anns are related to TF genes that are involved in response to salt stress, including SpAnn2 (DOF1), SpAnn4-2 (DOF1) and SpAnn6 (DOF1), SpAnn8 (DOF1, DOF2) in S. parvula and EsAnn3 (DOF2), EsAnn5 (DOF1) and EsAnn6 (DOF1, DOF2) in E. salsugineum.
Interestingly, in our investigation, GATA TFs were found in only one member of Ann genes promoter in the S. parvula (SpAnn2), but it was either greater than the total numbers of GATA in E. salsugineum or greater than that of A. thaliana. Gupta et al. [64] investigated GATA gene expression profiles in rice in response to salinity, drought and ABA treatments and found that some of the GATA genes have higher transcript levels in the salt tolerance genotype as compared to the salt sensitive variety. They mentioned that OsGATA1 and OsGATA10 might mediate abiotic stress responses and signaling. Zhao et al. [65], indicated that the SlGATA17-overexpressing tomatoes were more drought tolerant. Other studies also confirmed that GATA transcription factors promote abiotic tolerance [66,67]. It is noteworthy that Ann2 in both halophytes had six bHLH TF binding sites while A. thaliana did not. The bHLH TFs enhance plant's tolerance to abiotic stresses [68][69][70][71] for instance, in A. thaliana, AtbHLH028, AtbHLH92 and AtbHLH122 and also OsbHLH035, OsbHLH062 and OsbHLH068 in Oryza sativa are reported to participate in salt stress responses [72]. Based on TFs analysis, the expression patterns of selected SpAnn2 and also EsAnn2 were investigated, which revealed that they were upregulated in response to NaCl treatment. Several studies have also reported Ann2's role in abiotic stress responses [20,73].
According to RNA-seq data from Oh et al. [42], Ann6 in S. parvula compared to the A. thaliana homolog in basal (control) condition, shows 4.12-and 5.53-fold higher expression in roots and shoots, respectively (S8 Table). Besides, Harbaoui et al. [74] reported that overexpressed Arabidopsis plants with Triticum durum TdAnn6 improved salt and osmotic stress tolerance. In our investigation, NaCl treatment increased the expression level of the selected SpAnn6 gene in S. parvula shoots.
The results showed significant upregulation of EsAnn1, 5 and 7 in the roots of E. salsugineum seedlings which were treated with 300 mM NaCl and collected after 24 h [38] while in S. parvula's seedlings treated with 175 mM NaCl and collected after 3 h, 24 h, 48 h [39], candidate genes (Ann1,2 and 6) showed weak expression changes (range from -0.447 to +0.496). These differences in expressions could be attributed to the difference in NaCl concentration (175mM vs 300mM) and different seedling growth phase (4 days vs 30days after germination) in two halophytes. He et al. [9] found that most of Ann genes in group of Ann1, 2, 3 And 4 were upregulated under salinity and PEG stress in roots in Brassica napus. Some studies have shown that AtAnn1 and AtAnn2 regulated the growth and development of roots. Laohavisit et al. [15] demonstrated that root cell adaptation to salinity is impaired in the loss-of-function mutant of AtANN1. Wang et al. [75] also showed that AtANN1 and AtANN2 are important in postphloem sugar transport to the root tip, which influences photosynthetic rates in cotyledons. Ectopic expressed Ann6 of cotton in Arabidopsis made the root of transgenic plants longer due to the enlargement of root cells, without increasing the root cell number, through its interaction with actin 1 [76]. We also found that EsAnn1, 2 and 5 upregulated in E.salsugineum's leaves under salt stress treatment. These upregulations, as well as the expression pattern of three selected SpAnn genes in the shoot (SpAnn1, 2 and 6) support Anns' role in salt stress tolerance. The aforementioned expression profiles agree with cis-acting elements and TFs analysis.
Although upregulation of all Arabidopsis AtAnns except for Ann2 and Ann3 was observed in Arabidopsis salt induced AtAnns [77], we found that Ann2 in S. parvula was significantly upregulated in salt stress conditions. The divergent expression patterns of the two studied halophytes compared to A. thaliana as a glycophyte reveals that Ann genes in halophytes might change regulatory mechanisms that contribute to salt tolerance.

Conclusion
In this study, the S. parvula and E. salsugineum genomes were investigated in terms of Ann genes and ten and eight Ann genes were identified, respectively. Although the majority of homologous Ann gene pairs in S. parvula, E. salsugineum and A. thaliana had the same structure, conserved motifs and residues, the regulatory mechanisms (cis-acting elements and transcription factors) in halophytes are more salt responsive in compare to A. thaliana as a glycophyte. We chose three SpAnn genes (SpAnn1, 2 and 6) in S. parvula based on promoter region analysis as well as previous studies, which upregulated in response to NaCl treatment in the shoot. The expression patterns of E. salsugineum under salt stress supported Anns' role in salt stress tolerance. The divergent expression patterns of the investigated plants showed that SpAnn2 was upregulated under salt stress conditions, while AtAnn2 was not. Further investigation of the roles of candidate Ann genes in abiotic stresses, would be of great interest.
Supporting information S1 Table. Gene ontology (GO) terms annotation of five motifs in AtAnn, SpAnn and EsAnn proteins Identified by MEME tools.